Bacterial Cell Death Research Articles

Exposure of multidrug-resistant Klebsiella pneumoniae biofilms to 1,8-cineole leads to bacterial cell death and biomass disruption

Klebsiella pneumoniae is a common cause of health-care associated infections. The rise of antibiotic resistance and the ability to form biofilm among K. pneumoniae strains are two key factors associated with antibiotic treatment failure. The present study investigates the antibiofilm activity of 1,8-cineole against preformed biofilms of multidrug-resistant extended-spectrum β-lactamase-producing K. pneumoniae clinical isolates. To evaluate the antibiofilm activity, cellular viability was analyzed by colony-forming units counting and live/dead staining. In addition, biofilm biomass was evaluated by crystal violet and the biofilm matrix was stained with calcofluor white and observed by confocal laser scanning microscopy. A time- and concentration-dependent effect of the phytochemical over biofilm cell viability was observed revealing that 1% (v/v) 1,8-cineole during 1 h was the optimal treatment condition displaying a significant reduction of cell viability in the preformed biofilms (2.5–5.3 log cfu/cm2). Furthermore, confocal laser scanning microscopy after SYTO-9 and propidium iodide staining showed that 1,8-cineole was capable of killing bacteria throughout all layers of the biofilm. The compound also caused a biofilm disruption (30–62% biomass reduction determined by crystal violet staining) and a significant decrease in biofilm matrix density. Altogether, our results demonstrate that 1,8-cineole is a promising candidate as a novel antibiofilm agent against multidrug-resistant K. pneumoniae strains producing extended-spectrum β-lactamases, given its capability to disrupt the structure and to kill cells within the biofilm.

Ferrous sulfate-loaded hydrogel cures Staphylococcus aureus infection via facilitating a ferroptosis-like bacterial cell death in a mouse keratitis model

Hydrogels loaded with ampicillin, vancomycin or other antibiotics are one of the most widely used therapeutic agents for keratitis caused by Staphylococcus aureus. However, emergence of methicillin-resistant S. aureus (MRSA) makes infections harder to be treated by antibiotic-based hydrogels, urging the development of novel antibacterial materials. Inspired by mammalian ferroptosis, we determined the bactericidal effects of ferrous sulfate (FeSO4) on S. aureus, and evaluated the therapeutic potential of FeSO4-loaded hydrogel in a mouse keratitis model. The results showed that FeSO4 facilitated ferroptosis-like cell death in S. aureus with the key characteristics of reactive oxygen species (ROS) generation and lipid peroxidation. Notably, FeSO4 also efficiently killed persisters and MRSA, and eliminated biofilms of S. aureus. RNA profiles demonstrated that ferroptosis-related genes were significantly up-regulated, and the genes responsible for cell wall and cell membrane biosynthesis were down-regulated after exposure to Fe2+, supporting the occurrence of ferroptosis and cell lysis. We further prepared a FeSO4-loaded hydrogel by using hyaluronic acid (HA) and ascorbate. The FeSO4 hydrogel has the characteristics of injectability, self-healing, uniform distribution of Fe2+ in the three-dimensional gel structure, appropriate fluidity, high-water retention, high efficacy to kill MRSA, and excellent biocompatibility. In a mouse keratitis model, we showed that treatment of animals with FeSO4 hydrogel led to a rapid recovery of from keratitis, prevented the dissimilation of MRSA to the lung, and alleviated systemic inflammation, demonstrating the therapeutic potential of FeSO4 hydrogel. Taken together, our results indicated that FeSO4 hydrogel is a promising alternative to current antibiotics-dependent therapeutic materials for the treatment of infections by MRSA.

Dual antibacterial and anti-inflammatory efficacy of a chitosan-chondroitin sulfate-based in-situ forming wound dressing

None of the currently available wound dressings exhibit combined antibacterial and anti-inflammatory activity. Using polyelectrolyte complexation (PEC) between a cationic polysaccharide chitosan (CH) and an anionic glycosaminoglycan chondroitin sulfate (CS), we have developed a unique in-situ forming scaffold (CH-CS PEC), which develops at the wound site itself to influence the function of the wound bed cells. The current study demonstrated that CH-CS PEC could induce bacterial cell death through membrane pore formation and increased ROS production. Moreover, possibly due to its unique material properties including medium-soft viscoelasticity, porosity, and surface composition, CH-CS PEC could modulate macrophage function, increasing their phagocytic ability with low TNF-α and high IL-10 production. Faster wound closure and decreased CFU count was observed in an in-vivo infected wound model, with reduced NF-κB and increased VE-cadherin expression, indicating reduced inflammation and enhanced angiogenesis. In summary, this study exhibited that CH-CS PEC has substantial antibacterial and immunomodulatory properties.

The effect of nisin on the biofilm production, antimicrobial susceptibility and biofilm formation of Staphylococcus aureus and Pseudomonas aeruginosa

ObjectivesStaphylococcus aureus and Pseudomonas aeruginosa were the most common bacteria in nosocomial infections. Different bacteriocins are currently being studied as antibiotics or in conjunction with antibiotics as potential strategies to treat resistant infectious agents. The study aimed to determine nisin's effect on the biofilm production, antimicrobial susceptibility, and biofilm formation of S. aureus and P. aeruginosa.Materials and methodsThe experimental research tested two antibiotic-resistant isolates of S. aureus and P. aeruginosa strains. The experimental study tested two antibiotic-resistant isolates of S. aureus and P. aeruginosa strains. The MIC of bacteriocin nisin was determined using the micro broth dilution method, and crystal violet was used to assess the effect of bacteriocin on the biofilm. In addition, L929 cell culture was used to determine the effectiveness of bacteriocin on the isolate under similar cell conditions. Moreover, the MTT assay was used to and evaluate bacteriocin toxicity. In this study, the software Prism version 9 and Graph pad software were utilized.ResultsThe results of this study reveal that the nisin has different activities at different doses and is considered dose-dependent. At various times and doses, nisin inhibits biofilm formation in S. aureus, and P. aeruginosa isolates. Nisin also showed a decreasing survival of the isolates. Antibiotic-resistant bacteria can be made more vulnerable by nisin. Furthermore, nisin treatment affected the production of virulence factors such as hemolysins in S. aureus and had little or a negative effect on P. aeruginosa virulence factors. This medication stops S. aureus and P. aeruginosa from growing and causes bacterial cell damage.ConclusionsAntibacterial properties of nicin against S. aureus and P. aeruginosa were successfully studied. This bacteriocin stops S. aureus and P. aeruginosa from growing and causes bacterial cell damage or death. Damage to the membrane among the fundamental causes is reduced membrane potential and enzyme inactivation.

Aprotinin – Conjugated biocompatible porous nanocomposite for dentine remineralization and biofilm degradation

A nanocomposite (141 nm and +12.46 mV surface charge at pH 2.4) that could provide a single treatment for S. mutans-associated dental carries has been developed. The composite had six components, viz., aprotinin (Apr) that enzymatically inhibits plasmin activity of the bacterium, chitosan (Chi) that inhibits bacterial cell adhesion and biofilm formation, calcium phosphate (CaP) for remineralization of dentine, dexamethasone (DEX) for odontoblast differentiation, collagen (Cen) for crosslinking, and chlorogenic acid (CGA) for bacterial cell death. The structure of nanocomposite (HR SEM) consisted of interconnected pores and was amorphous post incorporation of chitosan. A sustained release of calcium and phosphate ions, lasting from 1 to 20 days at pH 4, an appropriate pH to combat demineralization, was observed, which is essential for remineralization. A 68.79% biofilm degradation was observed after 24 h of treatment, indicating good inhibition of the bacteria. While S. mutans grew well on denture resin specimen, the adhesion was reduced when the specimen was treated with the nanocomposite, indicating deterrence to biofilm formation. 5 Day hemolysis assay revealed red blood cell compatibility of up to 100 μg/25 μL, indicating that the nanocomposite was compatible with RBC. Against L132 cells, cytotoxicity was not observed up to 40 μg/mL for 72 h, a clear indication of non-cytotoxic character of the nanocomposite. CaP(NP)@Chi@CGA@DEX@Cen@Apr nanocomposite thus helps remineralization at cariogenic pH and biofilm degradation of S. mutans.

Phytochemical screening, antibacterial, anti-biofilm and quorum sensing inhibiting activity of Alpinia nigra leaf extract against infectious pathogen Pseudomonas aeruginosa PAO1

The recurrent cycles of several bacterial diseases are an endorsement of the formation of biofilms that are intractable to antibiotic therapy. Biofilm-associated diseases have affected millions of people worldwide and are unassailable due to frequent changes of surface antigens and gene expression. Therefore, there is a need to find out an alternative approach to cure biofilm infections. Plant-derived bioactive compounds and their derivative have shown a significant role in combatting bacterial infection. In this scenario, we performed phytochemical screening of ethyl acetate leaf extract (L-EtAc) of Alpina nigra and antibacterial, anti-biofilm activity towards Pseudomonas aeruginosa PAO1. Antibacterial and growth curve analysis of bacterial cells confirmed the significant bacterial cell death in L-EtAc treated sample. Relatively less biofilm formation was observed in the treated sample as revealed through fluorescence microscopy. Further, quorum-sensing inhibition was observed by applying a similar concentration of L-EtAc on Chromobacterium violaceum bacteria. The interaction between the biofilm-forming protein and phytochemical constituent was validated through in-silico analysis and further confirmed with qRT-PCR. The qRT-PCR data revealed reduced lasI, lasR, lasB, rhlI, rhlR and rhlA gene expression, which may be directly associated with biofilm and extracellular matrix formation. This study might be helpful for the development of new natural product for curing biofilm-associated disease.

Photodynamic antibacterial and antibiofilm activity of riboflavin against Xanthomonas oryzae pv oryzae: an ecofriendly strategy to combat bacterial leaf blight (BLB) rice disease.

Bacterial leaf blight (BLB), caused by Xanthomonas oryzae pv oryzae (Xoo), is one of the most damaging rice diseases, causing severe production losses depending on the rice variety. The purpose of this study was to develop an antibacterial photodynamic treatment (aPDT) using riboflavin for the treatment of BLB disease. Combining light and riboflavin (RF) therapy significantly reduced bacterial planktonic cells compared to RF alone. Photoactivated riboflavin also decreased biofilm biomass by reducing the number of viable sessile cells and the production of extracellular polymeric substances (EPS). Reactive oxygen species (ROS) levels in Xoo cells treated with photoactivated riboflavin were found to be significantly higher than in cells treated with riboflavin and light individually. Malondialdehyde (MDA) increased greatly in photoactivated riboflavin treated cells, indicating that severe oxidative damage was induced. Subsequently, a reduction in lactate dehydrogenase (LDH) activity in photoactivated riboflavin treated Xoo cells indicates that oxidative stress has disrupted the respiratory system, leading to bacterial cell death. In an ex vivo aPDT assay, photoactivated riboflavin successfully eradicated Xoo on the surface of rice leaves. Photoactivated riboflavin had no side effects on rice seed germination in subsequent trials, indicating that it is safe for agricultural applications. Therefore, all these findings suggest that aPDT is a potential alternative management strategy for BLB disease.

Antibacterial potency of cytocompatible chitosan-decorated biogenic silver nanoparticles and molecular insights towards cell-particle interaction

In the study, leaf extract of Carica papaya was utilized for the biogenic fabrication process of chitosan functionalized silver nanoparticles (Ag-Chito NPs). HRTEM analysis revealed that the fabricated Ag-Chito NPs was spherical in shape, with an average particle size of 13.31 (±0.07) nm. FTIR, UV–Vis, DLS, and other characterizations were also performed to analyze the diverse physicochemical properties of the particles. The antibacterial potency of the synthesized Ag-Chito NPs was tested against the two clinically isolated multidrug resistant uropathogenic bacterial strains, i.e. MLD 2 (Escherichia coli) and MLD 4 (Staphylococcus aureus) through MIC, MBC, time and concentration dependent killing kinetic assay, inhibition of biofilm formation assay, fluorescence and SEM imaging. Significantly, Ag-Chito NPs showed the highest sensitivity against the MLD 2 (MIC value of 12.5 μg/mL) strain, as compared to the MLD 4 (MIC value of 15 μg/mL) strain. From the hemolysis assay, it was revealed that Ag-Chito NPs exerted no significant toxicity up to 50 μg/mL against healthy human blood cells. Additionally, in silico analysis of chitosan (functionalized on the surface of AgNPs) and bacterial cell membrane protein also evidently suggested a strong interaction between Ag-Chito NPs and bacterial cells, which might be responsible for bacterial cell death.

Probiotic Microbes and Their Growth in Different Carriers (A Review)

Probiotics have been utilized as an option to treat problems brought on by safe and ecologically friendly pathogenic microorganisms in shrimp and fish farming operations. Probiotics are currently available in powder form (dry form). Using spray drying technology, dry probiotics are produced, with the finished result being a stable powder. In order to regulate the proliferation of harmful bacteria in the digestive tract, these bacteria use a competitive mechanism. Bacillus sp., Saccharomyces cereviceae, and Lactobacillus sp. are among the commonly researched probiotic microorganisms. Microorganisms are blended with a number of carriers, including maltodextrin, wheat flour, and rice flour. The carrier material's main goal is to increase storage process survival. Numerous studies have demonstrated that the viability and development of the probiotic microorganisms included in each carrier material varied. The enclosed substance exhibits highly varied microbial growth. The maximum number was found in the maltodextrin carrier material for the bacteria B. licheniformis and B. subtilis, the rice flour carrier for the bacteria L. brevis and L. curvatus, and the talc carrier for the bacterium L. bulgaricus. These findings suggest that, following encapsulation, probiotic populations and numbers can be maintained in all types of carrier materials. Specific carriers, like tapioca, skim milk, and activated carbon, revealed a greater microbial population than the typical medium. The microbial cells were compressed in the dry carrier material, which is why there was an increase of bacteria following the encapsulation procedure. Probiotic microbe counts increased by 40.7-90.1% for carriers made of activated carbon, 79-94.5% for those made of skim milk, and 31.7-95.7% for those made of tapioca flour. Bacterial cell death during the spray drying process of encapsulation could be the reason for the decline in the population of probiotic bacteria.

Formulasi dan Evaluasi Gel Nanopartikel Perak

Nanopartikel perak diketahui dapat menembus membran sel bakteri penyebab jerawat dan menyebabkan kerusakan struktural yang berakibat pada kematian sel bakteri. Nanopartikel perak perlu diformulasikan dalam bentuk gel agar memudahkan dalam penggunaannya. Penelitian ini dilakukan untuk mensintesis nanopartikel perak dan memformulasikannya dalam bentuk gel. Sintesis nanopartikel perak dilakukan menggunakan metode reduksi kimia dengan reduktor natrium sitrat dan penstabil gelatin. Nanopartikel perak kemudian diformulasikan dalam sediaan gel dengan variasi konsentrasi HPMC 7%, 10%, dan 15%. Hasil karakterisasi menunjukkan telah terbentuk nanopartikel perak berdasarkan panjang gelombang maksimal yang terbentuk yaitu 430,2 nm, absorbansi 0,297, ukuran partikel 157,73±15,03 nm, indeks polidispersitas 0,328±0,032, dan morfologi bulat. Berdasarkan hasil analisis, dapat disimpulkan bahwa formula 3 dengan konsentrasi HPMC 15% merupakan formula terbaik dengan sifat fisik berupa nilai viskositas 3931,75 ± 92,383 cps, diameter sebar 6,41 ± 0,06 cm dan daya sebar 22,78 ± 0,221 g.cm/detik, daya lekat 59,43 ± 1,158 detik, serta nilai pH 5,81 ± 0,04.

Exogenous metabolite feeding on altering antibiotic susceptibility in Gram-negative bacteria through metabolic modulation: a review.

The rise of antimicrobial resistance at an alarming rate is outpacing the development of new antibiotics. The worrisome trends of multidrug-resistant Gram-negative bacteria have enormously diminished existing antibiotic activity. Antibiotic treatments may inhibit bacterial growth or lead to induce bacterial cell death through disruption of bacterial metabolism directly or indirectly. In light of this, it is imperative to have a thorough understanding of the relationship of bacterial metabolism with antimicrobial activity and leverage the underlying principle towards development of novel and effective antimicrobial therapies. Herein, we explore studies on metabolic analyses of Gram-negative pathogens upon antibiotic treatment. Metabolomic studies revealed that antibiotic therapy caused changes of metabolites abundance and perturbed the bacterial metabolism. Following this line of thought, addition of exogenous metabolite has been employed in in vitro, in vivo and in silico studies to activate the bacterial metabolism and thus potentiate the antibiotic activity. Exogenous metabolites were discovered to cause metabolic modulation through activation of central carbon metabolism and cellular respiration, stimulation of proton motive force, increase of membrane potential, improvement of host immune protection, alteration of gut microbiome, and eventually facilitating antibiotic killing. The use of metabolites as antimicrobial adjuvants may be a promising approach in the fight against multidrug-resistant pathogens.

Anti-Inflammatory Effects of Miang Bean Leaves (Mucuna pruriens)

Inflammation is a serious health problem that needs treatment. The use of steroids and non-steroidal anti-inflammatory drugs (NSAIDs) can actually relieve inflammatory reactions well, but their long-term use can have many side effects and are uneconomical. Therefore, the use of natural ingredients that are effective and economical is needed. One of the plants that are considered anti-inflammatory is the leaves of miang beans (Mucuna pruriens). The purpose of compiling this narrative review is to analyze the phytochemical content of miang beans leaves and its mechanisms for inflammatory reactions. The narrative study of this preparation was carried out using the literature study method from August to September 2020. The phytochemical content of miang bean leaves are alkaloids, flavonoids, saponins, cyanogenic glycosides, and tannins. Phytochemicals that have a direct anti-inflammatory effect are alkaloids, flavonoids, saponins, and tannins. Alkaloids are antibacterial. If there are bacteria that cause inflammation, the alkaloids will damage the bacterial peptidoglycan cell walls, thus causing bacterial cell death and preventable inflammatory reactions. Flavonoids have anti-inflammatory effects by inhibiting macrophages from producing NO (nitric oxide) and inhibiting the cyclooxygenase pathway. Saponins affect the inflammatory reaction by inhibiting the lipoxygenase pathway and inhibiting the release of inflammatory mediators. Meanwhile, tannins affect the inflammatory reaction by inhibiting macrophages from producing ROS (Reactive Oxygen Species). Potential which is fatal because it can turn into cyanide acid. However, the content of these cyanogenic glycosides can be minimized by drying at low temperatures. So, mung bean leaves are effective as anti-inflammatory drugs because the alkaloids, flavonoids, saponins, and tannins they contain are anti-inflammatory.

Cell Death in Escherichia coli: Incomplete Base Excision Repair under Depletion of DapB and Dxr Proteins.

ABSTRACTThe generation of reactive oxygen species (ROS) within the cell is a significantly shared aspect of bacterial cell death against different stress conditions. The main cell death mechanism due to the generation of reactive oxygen species is then the incomplete base excision repair (BER) in response to oxidized nucleotides. In their recent article in mBio, C. C. Gruber, V. M. P. Babu, K. Livingston, H. Joisher, and G. C. Walker (mBio 13[1]:e03756-21, 2022) report two new stress conditions regarding the depletion of DapB and Dxr, which indeed cause similar mechanisms for cell death. These two stress conditions trigger highly distinctive stress response mechanisms within the cell, but the ultimate cell death mechanism is a result of a shared process. These findings prove that the disturbance in the homeostasis of cells under a variety of different stresses initiates cell death mechanisms through the production of ROS, generation of 8-oxo-dG and the incomplete BER.

Secondary Ammonium-Based Hyperbranched Poly(amidoamine) with Excellent Membrane-Active Property for Multidrug-Resistant Bacterial Infection.

With the rapid emergence of microbial infections induced by "superbugs", public health and the global economy are threatened by the lack of effective and biocompatible antibacterial agents. Herein, we systematically design a series of secondary ammonium-based hyperbranched poly(amidoamine) (SAHBP) with different alkyl chain lengths for probing high-efficacy antibacterial agents. SAHBP modified with alkyl tails at the hyperbranched core could efficiently kill Escherichia coli and Staphylococcus aureus, two types of clinically important bacteria worldwide. The best SAHBP with 12-carbon-long alkyl tails (SAHBP-12) also showed high activity against problematic multidrug-resistant bacteria, including Pseudomonas aeruginosa and methicillin-resistant S. aureus (MRSA). Based on ζ potential, isothermal titration microcalorimetry (ITC), and membrane integrity assays, it is found that SAHBP-12 could attach to the cell membrane via electrostatic adsorption and hydrophobic interactions, following which the integrity of the bacterial cell wall and the cell membrane is disrupted, resulting in severe cell membrane damage and the leakage of cytoplasmic contents, finally causing bacterial cell death. Impressively, benefiting from excellent membrane-active property, SAHBP-12 exhibited robust therapeutic efficacy in MRSA-infected mice wounds. Moreover, SAHBP-12 also showed excellent biosafety in vitro and in vivo, which undoubtedly distinguished it as a potent weapon in combating the growing threat of problematic multidrug-resistant bacterial infections.

Otilonium bromide boosts antimicrobial activities of colistin against Gram-negative pathogens and their persisters

Colistin is the last-line antibiotic against Gram-negative pathogens. Here we identify an FDA-approved drug, Otilonium bromide (Ob), which restores the activity of colistin against colistin-resistant Gram-negative bacteria in vitro and in a mouse infection model. Ob also reduces the colistin dosage required for effective treatment of infections caused by colistin-susceptible bacteria, thereby reducing the toxicity of the drug regimen. Furthermore, Ob acts synergistically with colistin in eradicating multidrug-tolerant persisters of Gram-negative bacteria in vitro. Functional studies and microscopy assays confirm that the synergistic antimicrobial effect exhibited by the Ob and colistin involves permeabilizing the bacterial cell membrane, dissipating proton motive force and suppressing efflux pumps, resulting in membrane damages, cytosol leakage and eventually bacterial cell death. Our findings suggest that Ob is a colistin adjuvant which can restore the clinical value of colistin in combating life-threatening, multidrug resistant Gram-negative pathogens.

Synthesis of Rutin loaded nanomagnesia as a smart nanoformulation with significant antibacterial and antioxidant properties

Nowadays synthesis of hybrid nanomaterials is of interest to researchers due to their profound antioxidant and antibacterial properties. Here, at first we prepared amine functionalized positively charged MgO nanoparticles (MgO-NH2) via microwave assisted heating followed by annealing at 400 °C for 5 h, and conjugated thereafter with a natural flavonoid, Rutin to form a bioactive nanohybrid, MgO-NH2-Rutin. The nearly spherically shaped nanohybrids with size ∼35–40 nm showed drug loading content of ∼14 % with a sustained release pattern at pH 7.4. The nanohybrids exhibited superior antioxidant properties in cell free system (DPPH, hydrogen peroxide and superoxide) as well as in intracellular condition, and showed very little cytotoxicity upto a concentration of 100 μg/mL in normal kidney epithelial cells. Besides, MgO-NH2-Rutin also triggered a significant growth inhibition against both gram positive (S. aureus) & gram negative (E. coli) bacteria. Mechanistic studies showed that the nanohybrid caused bacterial cell death via oxidative stress, DNA degradation and cell membrane disruption. Therefore, our research highlighted the use of MgO nanoparticles for the synthesis of MgO-NH2-Rutin nanohybrid, which could enhance the pharmacological actions of hydrophobic drug molecules and exhibit synergistic antioxidant and antibacterial activities.

Immersed Boundary Approach to Biofilm Spread on Surfaces

We propose a computational framework to study the growth and spread of bacterial biofilms on interfaces, as well as the action of antibiotics on them. Bacterial membranes are represented by boundaries immersed in a fluid matrix and subject to interaction forces. Growth, division and death of bacterial cells follow dynamic energy budget rules, in response to variations in environmental concentrations of nutrients, toxicants and substances released by the cells. In this way, we create, destroy and enlarge boundaries, either spherical or rod-like. Appropriate forces represent details of the interaction between cells, and the interaction with the environment. Numerical simulations illustrate the evolution of top views and diametral slices of small biofilm seeds, as well as the action of antibiotics. We show that cocktails of antibiotics targeting active and dormant cells can entirely eradicate a biofilm.

Colonic MUC2 Mucin Is Stabilized By FCGBP To Resist Salmonella Enterica Invasion In Goblet Cells

MUC2 mucin produced by goblet cells form the colonic mucus bilayer as the first line of innate defense against pathogen invasion by forming a physical barrier between the lumen and the underlying single layer of mucosal epithelial cells. Goblet cells also produce a variety of proteins that are associated with the mucus layer. Of these proteins, FCGBP is of significant interest due to its structural similarities to MUC2 mucin with unknown functions. In this study, we elucidated the putative functions of FCGBP embedded in MUC2 mucin in response to Salmonella enterica adherence and invasion in human colonic goblet cells. We hypothesis that FCGBP is coordinately produced with MUC2 mucin to play an integral role in forming the protective mucus layer in innate host defense. The aims of the study are: 1) to determine if FCGBP alters the structural integrity of the mucus layer and 2) to quantify structural alterations of the mucus layer in S. enterica adherence and invasion of goblet cells.To investigate whether FCGBP impaired mucus barrier functions, two cell types were investigated: Wildtype LS174T (WT) MUC2 mucus‐producing goblet cells and LS174T cells with a missense mutation in FCGBP (FCGBP MS). To determine if FCGBP MS led to loss in barrier function in the mucus layer, S. enterica adherence and invasion, and 0.2, 1, and 2 µM fluorescent bead penetrability inoculated on WT and FCGBP MS monolayers were quantified. MUC2 and FCGBP mRNA and protein expression induced by S. enterica in WT and FCGBP MS goblet cells were analysed by RT‐PCR and Western blotting. Cell killing by S. enterica was determined by LDH assay. S. enterica induced pro‐inflammatory cytokines responses were analyzed by RT‐PCR and human focus 15‐plex cytokine and chemokine array.FCGBP MS cells exhibited significant loss in MUC2 mucus barrier function as quantified by fluorescent beads penetrability further towards the epithelial cell surface temporally as compared to WT cells independent of bead size. Adherence of live S. enterica was significantly increased in FCGBP MS but not WT cells and induced robust MUC2 mRNA expression and mucus secretion in a time‐dependent manner. Similarly, S. enterica elicited high expression of pro‐inflammatory cytokine mRNA and protein release in FCGBP MS as compared to WT cells. FCGBP MS were readily invaded by S. enterica that resulted in increased cell death as compared to WT cells.More S. enterica adhered, invaded, and killed FCGBP MS as compared to WT cells. Fluorescent beads penetrated the mucus layer closer to the cell surface in FCGBP MS cells. In WT goblet cells, FCGBP and MUC2 were coordinately upregulated in response to S. enterica. The concurrent increase in pro‐inflammatory cytokine expression in FCGBP MS cells in response to S. enterica suggests that bacteria directly interacted with the cell surface suggesting an impaired protective mucus layer. The overall trend for increased bacterial invasion, pro‐inflammatory response, and cell death in FCGBP MS demonstrates that FCGBP was critical in providing structural integrity of the mucus layer.

Antibacterial activity of colloidal copper nanoparticles against Gram-negative (Escherichia coli and Proteus vulgaris) bacteria.

Antibacterial activities of as-synthesized nanoparticles have gained attention in past few years due to rapid phylogenesis of pathogens developing multi-drug resistance (MDR). Antibacterial activity of copper nanoparticles (CuNPs) on surrogate pathogenic Gram-negative bacteria Escherichia coli (MTCC no. 739) and Proteus vulgaris (MTCC no. 426) was evaluated under culture conditions. Three sets of colloidal CuNPs were synthesized by chemical reduction method with per batch yield of 0·2, 0·3 and 0·4 g. As-synthesized CuNPs possess identical plasmonic properties and have similar hydrodynamic particle sizes (11-14 nm). Antibacterial activities of CuNPs were evaluated by minimum inhibitory concentration (MIC) and minimum bactericidal concentration (MBC) tests, cytoplasmic leakage and reactive oxygen species (ROS) assays. MIC and MBC tests revealed dose dependence bactericidal action. Growth curves of E. coli show faster growth inhibition along with higher cytoplasmic leakage than that of P. vulgaris. This might be because of increased membrane permeability of E. coli. CuNP-microorganism interaction induces oxidative stress generated by ROS. Leakage of cytoplasmic components, loss of membrane permeability and ROS generation are the primary causes of CuNP-induced bacterial cell death. As-synthesized CuNPs exhibiting promising antibacterial activities and could be a promising candidate for novel antibacterial agents.

Lacritin Bactericidal Peptide N‐104 Targeting of Inner Membrane Transporters FeoB and PotH (SpuG) Individually Absent in N‐104 Resistant Strains of Human Opportunistic Pathogen, P. aeruginosa PA14

Through an unbiased screen of the E. coli Keio collection of 3,985 nonessential gene knockouts, we have discovered that inner membrane putrescine and ferrous iron transporters PotH and FeoB each play roles in the bactericidal mechanism of tear lacritin peptide ‘N‐104’ ‐ possibly involving outer‐membrane translocation by lipoprotein YaiW with which it binds and when absent confers N‐104 resistance. The results were confirmed in the human opportunistic pathogen, P. aeruginosa PA14, and in a robust C. elegans‐based liquid infection assay. Does periplasmic N‐104 directly or indirectly target PotH and/or FeoB? His‐tagged FeoB (P. aeruginosa) and PotH (E. coli) extracted in lysis buffer containing 2% Dodecyl‐b‐D‐maltopyranoside were purified on Ni‐Sepharose columns, concentrated on 50 kDa (FeoB) or 30 kDa (PotH) Amicon cutoff membranes, and then added in equal amounts in 150 mM NaCl column buffer to SulfoLink® immobilized Cys‐N‐104 or to negative control Cys‐C‐95 columns. After overnight incubation at 4°C and collection of the flow through, columns were washed with 25 column volumes of 150 mM KCl before elution via a gradient of column buffer containing 300, 500, or 1000 mM KCl. Whereas FeoB and PotH were detected only in the flow through off of C‐95, 500 mM KCl was required for elution off of N‐104. PotH inwardly transports putrescine, the predominant polyamine in bacterial cells (10 ‐ 30 mM) that is essential for optimal bacterial cell growth and proliferation. FeoB is the major mediator of ferrous iron uptake in P. aeruginosa, E. coli, H. pylori, and some other species, and is a well‐recognized virulence factor. Given that none of those proteins are essential for the bacterial survival, we hypothesize that N‐104 might either disturb putrescine or ferrous iron regulation, or employ each to trigger programmed cell death via uncharacterized YbdM predicted to have transcription regulatory region activity also discovered to bind N‐104, and when absent confers N‐104 resistance. This study uncovers new virulence factors, and a novel method of bacterial cell death by a natural peptide component of human tears and apparently of plasma.